Taxol enhancer compounds

ABSTRACT

Disclosed is a compound represented by the Structural Formula (I): 
                 
         Y is a covalent bond, a phenylene group or a substituted or unsubstituted straight chained hydrocarbyl group. In addition, Y, taken together with both &gt;C═Z groups to which it is bonded, is a substituted or unsubstituted aromatic group. Preferably, Y is a covalent bond or —C(R 7 R 8 )—.   R 1  and R 2  are independently an aryl group or a substituted aryl group, R 3  and R 4  are independently —H, an aliphatic group, a substituted aliphatic group, an aryl group or a substituted aryl group.   R 5 -R 6  are independently —H, an aliphatic group, a substituted aliphatic group, an aryl group or a substituted aryl group.   R 7  and R 8  are each independently —H, an aliphatic or substituted aliphatic group, or R 7  is —H and R 8  is a substituted or unsubstituted aryl group, or, R 7  and R 8 , taken together, are a C2-C6 substituted or unsubstituted alkylene group.   Z is ═O or ═S.       

     Also disclosed are pharmaceutical compositions comprising the compound of the present invention and a pharmaceutically acceptable carrier or diluent. Also disclosed is a method of treating a subject with cancer by administering to the subject a compound of Structural Formula (I) in combination with taxol or an analog of taxol.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 10/193,075,filed Jul. 10, 2002, which claims the benefit of U.S. ProvisionalApplication No. 60/304,252, filed Jul. 10, 2001, and U.S. ProvisionalApplication No. 60/361,946, filed Mar. 6, 2002. This application is alsoa continuation-in-part of U.S. Ser. No. 10/193,639, filed Jul. 10, 2002,which claims the benefit of U.S. Provisional Application No. 60/304,252,filed Jul. 10, 2001, and U.S. Provisional Application No. 60/361,936,filed Mar. 6, 2002. The entire teachings of these applications areincorporated herein by reference.

BACKGROUND OF THE INVENTION

Many new drugs are now available to be used by oncologists in treatingpatients with cancer. Often, tumors are more responsive to treatmentwhen anti-cancer drugs are administered in combination to the patientthan when the same drugs are administered individually and sequentially.One advantage of this approach is that the anti-cancer agents often actsynergistically because the tumors cells are attacked simultaneouslywith agents having multiple modes of action. Thus, it is often possibleto achieve more rapid reductions in tumor size by administering thesedrugs in combination. Another advantage of combination chemotherapy isthat tumors are more likely to be eradicated completely and are lesslikely to develop resistance to the anti-cancer drugs being used totreat the patient.

One serious limitation of combination chemotherapy is that anti-canceragents generally have severe side effects, even when administeredindividually. For example, the well known anti-cancer agent taxol causesneutroperia, neuropathy, mucositis, anemia, thrombocytopenia,bradycardia, diarrhea and nausea. Unfortunately, the toxicity ofanti-cancer agents is generally additive when the drugs are administeredin combination. As result, certain types of anti-cancer drugs aregenerally not combined. The combined toxic side-effects of thoseanti-cancer drugs that are administered simultaneously can place severelimitations on the quantities that can be used in combination. Often, itis not possible to use enough of the combination therapy to achieve thedesired synergistic effects. Therefore, there is an urgent need foragents which can enhance the desirable tumor attacking properties ofanti-cancer agents without further increasing their undesirableside-effects.

SUMMARY OF THE INVENTION

It has now been found that certain bis[thio-hydrazide amide] compoundssignificantly enhance the anti-cancer activity of taxol. For example,Compound (1) was used in combination with taxol (Paclitaxel) to treattumors induced in nude mice from the human breast tumor cell lineMDA-435. The tumor volume was about five fold less after 24 days oftreatment in mice which had been administered 5 mg/kg of taxol and 25mg/kg of Compound (1) than in mice which had only been administered 5mg/kg of taxol or in mice which had only been administered 50 mg/kg ofCompound (1) (Example 13). These results are shown graphically in FIG.1. The structure of Compound (1) is shown below:

It has also been found that these bis[thio-hydrazide amide] compoundshave minimal toxic side effects. For example, the mice treated withtaxol and Compound (1) showed little if any weight loss over thetreatment period (see FIG. 2). Based on these results, novel compoundswhich enhance the anti-cancer activity of taxol, pharmaceuticalcompositions comprising these compounds and methods of treating asubject with cancer are disclosed herein.

One embodiment of the present invention is a compound represented by theStructural Formula (I):

-   -   Y is a covalent bond, a phenylene group or a substituted or        unsubstituted straight chained hydrocarbyl group. In addition,        Y, taken together with both >C═Z groups to which it is bonded,        is a substituted or unsubstituted aromatic group. Preferably, Y        is a covalent bond or —C(R₇R₈)—.    -   R₁ and R₂ are independently an aryl group or a substituted aryl        group, R₃ and R₄ are independently —H, an aliphatic group, a        substituted aliphatic group, an aryl group or a substituted aryl        group.    -   R₅-R₆ are independently —H, an aliphatic group, a substituted        aliphatic group, an aryl group or a substituted aryl group.    -   R₇ and R₈ are each independently —H, an aliphatic or substituted        aliphatic group, or R₇ is —H and R₈ is a substituted or        unsubstituted aryl group, or, R₇ and R₈, taken together, are a        C2-C6 substituted or unsubstituted alkylene group.    -   Z is ═O or ═S.

In one aspect, R₁ and R₂ in the compound represented by StructuralFormula (I) are not both phenyl when Y is —C(R₇R₈)—, R₃ and R₄ are bothphenyl and R₅-R₈ are all —H.

Another embodiment of the present invention is a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier or diluentand a compound represented by Structural Formula (I). Preferably, thepharmaceutical composition comprises an effective concentration of thecompound.

Yet another embodiment of the present invention is a method of treatinga subject with cancer. The method comprises administering to the subjectan effective amount of taxol or a taxol analog and an effective amountof a compound represented by Structural Formula (I).

Yet another embodiment of the present invention is a method of treatinga subject with cancer. The method comprises the step of administering tothe subject an effective amount of a compound selected from:

or a pharmaceutically acceptable salt thereof.

The disclosed compounds increase the anti-cancer activity of taxol andtaxol analogs. In addition, these compounds have minimal toxicside-effects. Consequently, it is possible to increase the effectivenessof taxol and analogs thereof when used in combination with the disclosedcompounds, even when approaching the highest tolerated doses of taxol.Thus, it is expected that combination therapy with the compounds of thepresent invention will provide improved clinical outcomes for patientswith cancers that are being treated with taxol. By co-administering thedisclosed compounds with taxol, it is also possible to achieve the sametherapeutic effectiveness previously achieved with higher doses oftaxol, thereby reducing the side-effects and improving the quality oflife for the patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the average tumor volume in milliliters overtime (in days) in nude mice treated with vehicle; Compound (1) (50mg/kg); Paclitaxel (5 mg/kg); Compound (1) (25 mg/kg) and Paclitaxel (5mg/kg); or Compound (1) (50 mg/kg) and Paclitaxel (5 mg/kg). The tumorswere generated from the human breast tumor cell line MDA-435.

FIG. 2 is a graph showing the percent weight change over time in nudemice treated with vehicle; Compound (1) (50 mg/kg); Paclitaxel (5mg/kg); Compound (1) (25 mg/kg) and Paclitaxel (5 mg/kg); or Compound(1) (50 mg/kg) and Paclitaxel (5 mg/kg). The mice were being treated fortumors generated from the human breast tumor cell line MDA-435.

FIG. 3 is the structure of taxol (Paclitaxel)

FIG. 4 is the structure of taxotere (Docetaxel)

FIGS. 5-25 are each the structure of a taxol analog.

FIG. 26 is the structure of a polymer comprising a taxol analog grouppendent from the polymer backbone. The polymer is a terpolymer of thethree monomer units shown.

FIG. 27 is a graph showing the average tumor volume in milliliters overtime (in days) in nude mice treated with vehicle (●); Compound (1) (25mg/kg) (♦); Paclitaxel (15 mg/kg) (▪); or Compound (1) (25 mg/kg) andPaclitaxel (15 mg/kg) (□). The tumors were generated from the humanbreast tumor cell line MDA-435.

FIG. 28 is a graph showing the percent weight change over time in nudemice treated with vehicle (●); Compound (1) (25 mg/kg) (♦); Paclitaxel(15 mg/kg) (▪); or Compound (1) (25 mg/kg) and Paclitaxel (15 mg/kg)(□). The mice were being treated for tumors generated from the humanbreast tumor cell line MDA-435.

DETAILED DESCRIPTION OF THE INVENTION

In a first preferred embodiment, Y in Structural Formula (I), takentogether with both >C═Z groups to which it is bonded, is a substitutedor unsubstituted arylene group and the compound is represented byStructural Formula (II):

-   -   R₁-R₆ in Structural Formula (II) are as described in Structural        Formula (I). Ar is a substituted or unsubstituted arylene group.        Preferably, Ar is a nitrogen-containing heteroarylene group.        Examples are shown below:        Ring A is substituted or unsubstituted.

In a second preferred embodiment, Y in Structural Formula (I) is acovalent bond or a substituted or unsubstituted straight chainedhydrocarbyl group. R₇ and R₈ are as described for Structural Formula(I). Preferably, Y is a covalent bond, —C(R₇R₈)—, —(CH₂CH₂)—,trans-(CH═CH)—, cis-(CH═CH)—, —(CC)— or a 1,4-phenylene group. Even morepreferably, Y is a covalent bond or —C(R₇R₈)—.

In a third preferred embodiment, Y in Structural Formula (I) is acovalent bond or —C(R₇R₈)— and the compound of the present invention isrepresented by Structural Formula (III):

-   -   R₁-R₈ are as described for Structural Formula (I). Y′ is a        covalent bond or —C(R₇R₈)—. Preferably, R₇ and R₈ are both        methyl; R₇ and R₈, taken together, are propylene or butylene; or        R₇ is —H and R₈ is lower alkyl (preferably methyl), thienyl,        phenyl, benzyl, or amino.

In a more preferred embodiment, R₅-R₈ in Structural Formula (III) are —Hand the compound is represented by Structural Formula (IV):

-   -   R₁-R₄ in Structural Formula (IV) are as described in Structural        Formula (I). Y″ is a covalent bond or —CH₂—.

In a first example of a compound represented by Structural Formula (IV),R₃ and R₄ are both a substituted or unsubstituted aliphatic group,preferably both a substituted or unsubstituted lower alkyl group andmore preferably both a methyl group or ethyl. When R₃ and R₄ inStructural Formula (IV) are both a substituted or unsubstitutedaliphatic group, then R₁ and R₂ are preferably both a substituted orunsubstituted aryl group (e.g., a substituted or unsubstitutedheteroaryl group, a substituted or unsubstituted phenyl group, or aphenyl group with at least one substituent other than an aliphaticgroup).

In a second example of a compound represented by Structural Formula(IV), R₃ and R₄ are both a substituted or unsubstituted heteroarylgroup. When R₃ and R₄ in Structural Formula (IV) are both a substitutedor unsubstituted heteroaryl group, then R₁ and R₂ are preferablyboth: 1) a substituted or unsubstituted phenyl group; or 2) asubstituted or unsubstituted heteroaryl group.

In a third example of a compound represented by Structural Formula (IV),R₃ and R₄ are both a substituted or unsubstituted phenyl group (e.g., aphenyl group substituted with at least one group other than an aliphaticgroup). When R₃ and R₄ in Structural Formula (IV) are both a substitutedor unsubstituted phenyl group, then R₁ and R₂ are preferably both: 1) asubstituted or unsubstituted phenyl group; or 2) a substituted orunsubstituted heteroaryl group.

In a fourth example of a compound represented by Structural Formula(IV), R₁ and R₂ are both a substituted or unsubstituted aryl group(e.g., a substituted or unsubstituted heteroaryl group, a substituted orunsubstituted phenyl group or a phenyl group substituted with at leastone group other than an aliphatic group). More preferably, R₃ and R₄ areboth methyl and the remainder of the variables are as described above.

In a fourth preferred embodiment, the compound of the present inventionis represented by Structural Formula (III), wherein at least one ofR₁-R₄ is a heteroaryl group, a substituted heteroaryl group, or a phenylgroup substituted with at least one group other than an aliphatic group.Preferably, R₅-R₈ are all —H.

The following are specific examples of compounds represented byStructural Formula (IV): R₁ and R₂ are both phenyl, and R₃ and R₄ areboth o-CH₃-phenyl; R₁ and R₂ are both o-CH₃C(O)O-phenyl, and R₃ and R₄are phenyl; R₁ and R₂ are both phenyl, and R₃ and R₄ are both methyl; R₁and R₂ are both phenyl, and R₃ and R₄ are both ethyl;

-   -   R₁ and R₂ are both phenyl, and R₃ and R₄ are both n-propyl; R₁        and R₂ are both p-cyanophenyl, and R₃ and R₄ are both methyl; R₁        and R₂ are both p-nitro phenyl, and R₃ and R₄ are both methyl;        R₁ and R₂ are both 2,5-dimethoxyphenyl, and R₃ and R₄ are both        methyl; R₁ and R₂ are both phenyl, and R₃ and R₄ are both        n-butyl; R₁ and R₂ are both p-chlorophenyl, and R₃ and R₄ are        both methyl;    -   R₁ and R₂ are both 3-nitrophenyl, and R₃ and R₄ are both methyl;        R₁ and R₂ are both 3-cyanophenyl, and R₃ and R₄ are both methyl;        R₁ and R₂ are both 3-fluorophenyl, and R₃ and R₄ are both        methyl; R₁ and R₂ are both 2-furanyl, and R₃ and R₄ are both        phenyl;    -   R₁ and R₂ are both 2-methoxyphenyl, and R₃ and R₄ are both        methyl; R₁ and R₂ are both 3-methoxyphenyl, and R₃ and R₄ are        both methyl; R₁ and R₂ are both 2,3-dimethoxyphenyl, and R₃ and        R₄ are both methyl; R₁ and R₂ are both 2-methoxy-5-chlorophenyl,        and R₃ and R₄ are both ethyl; R₁ and R₂ are both        2,5-difluorophenyl, and R₃ and R₄ are both methyl; R₁ and R₂ are        both 2,5-dichlorophenyl, and R₃ and R₄ are both methyl; R₁ and        R₂ are both 2,5-dimethylphenyl, and R₃ and R₄ are both methyl;    -   R₁ and R₂ are both 2-methoxy-5-chlorophenyl, and R₃ and R₄ are        both methyl;    -   R₁ and R₂ are both 3,6-dimethoxyphenyl, and R₃ and R₄ are both        methyl; R₁ and R₂ are both phenyl, and R₃ and R₄ are both        2-ethylphenyl; R₁ and R₂ are both 2-methyl-5-pyridyl, and R₃ and        R₄ are both methyl; or R₁ is phenyl; R₂ is 2,5-dimethoxyphenyl,        and R₃ and R₄ are both methyl.

In a fifth preferred embodiment, Y in Structural Formula (I) is—C(R₇R₈)— and R₅ and R₆ are both —H. When Y is a covalent bond or—CR₇R₈— and R₅ and R₆ are both —H, the compound of the present inventionis represented by Structural Formula (V):

-   -   R₁-R₄, R₇ and R₈ are as described for Structural Formula (I) and        Y′ is a covalent bond or —CR₇R₈—. R₇ and R₈ are the same or        different. Preferably, R₇ and R₈ are both methyl;    -   R₇ and R₈, taken together, are propylene or butylene; or R₇ is        —H and R₈ is lower alkyl (preferably methyl), thienyl, phenyl or        benzyl.

In one example of a compound represented by Structural Formula (V), R₁and R₂ are both aryl or substituted aryl groups and R₃ and R₄ are both alower alkyl group or a substituted lower alkyl group; preferably, R₁ andR₂ are both aryl or substituted aryl groups, R₃ and R₄ are both methylor ethyl, R₇ is —H and R₈ is —H or methyl. In another example of acompound represented by Structural Formula (V), R₁ and R₂ are bothphenyl or substituted phenyl and R₃ and R₄ are both methyl, ethyl,phenyl, or thienyl. When R₁ and R₂ are both phenyl or substituted phenyland R₃ and R₄ are both methyl, ethyl, phenyl, or thienyl, thenpreferably R₇ and R₈, taken together, are propylene or butylenes. In yetanother example of a compound represented by Structural Formula (V), Y′is a covalent bond or —CR₇R₈—; R₁ and R₂ are both a substituted orunsubstituted aryl group; R₃ and R₄ are both —H, methyl or ethyl; and R₇is —H and R₈ is —H or methyl.

The following are specific examples of compounds represented byStructural Formula (V): R₁ and R₂ are both phenyl; R₃ and R₄ are bothmethyl; R₇ is —H, and R₈ is ethyl; R₁ and R₂ are both phenyl; R₃ and R₄are both phenyl, and R₇ and R₈ are both methyl; R₁ and R₂ are both2-thienyl; R₃ and R₄ are both phenyl, and R₇ and R₈ are both methyl; R₁and R₂ are both 4-cyanophenyl; R₃ and R₄ are both methyl; R₇ is —H, andR₈ is methyl; R₁ and R₂ are both phenyl; R₃ and R₄ are both methyl; R₇is —H, and R₈ is methyl; R₁ and R₂ are both phenyl; R₃ and R₄ are bothmethyl; R₇ is —H, and R₈ is benzyl; R₁ and R₂ are both phenyl; R₃ and R₄are both methyl; R₇ is —H, and R₈ is ethyl; R₁ and R₂ are both phenyl;R₃ and R₄ are both ethyl; R₇ is —H, and R₈ is n-butyl; R₁ and R₂ areboth 2,5-dimethoxyphenyl; R₃ and R₄ are both methyl; R₇ is —H, and R₈ ismethyl; R₁ and R₂ are both phenyl; R₃ and R₄ are both methyl; R₇ is —H,and R₈ is iso-propyl; R₁ and R₂ are both 3-nitrophenyl; R₃ and R₄ areboth methyl; R₇ is —H, and R₈ is methyl; R₁ and R₂ are both4-chlorophenyl; R₃ and R₄ are both methyl; R₇ is —H, and R₈ is methyl;R₁ and R₂ are both phenyl; R₃ and R₄ are both methyl; R₇ is —H, and R₈is 3-thienyl; R₁ and R₂ are both phenyl; R₃ and R₄ are both methyl, andR₇ and R₈, taken together, are propylene; R₁ and R₂ are both2,3-dimethoxyphenyl; R₃ and R₄ are both methyl; R₇ is —H, and R₈ ismethyl; R₁ and R₂ are both 2-chloro-5-methoxy phenyl; R₃ and R₄ are bothmethyl; R₇ is —H, and R₈ is methyl; R₁ and R₂ are both2,5-difluorophenyl; R₃ and R₄ are both methyl; R₇ is —H, and R₈ ismethyl; R₁ and R₂ are both 2,5-dichlorophenyl; R₃ and R₄ are bothmethyl; R₇ is —H, and R₈ is methyl; R₁ and R₂ are both2,6-dimethoxyphenyl; R₃ and R₄ are both methyl; R₇ is —H, and R₈ ismethyl; R₁ and R₂ are both 2,5-dimethylphenyl; R₃ and R₄ are bothmethyl; R₇ is —H, and R₈ is methyl; R₁ and R₂ are both2,5-dimethoxyphenyl; R₃ and R₄ are both ethyl; R₇ is —H, and R₈ ismethyl, and R₁ and R₂ are both 2,5-diethoxyphenyl; R₃ and R₄ are bothmethyl; R₇ is —H, and R₈ is methyl.

In a sixth preferred embodiment, Y in Structural Formula (I) is acovalent bond or —CH₂—. When Y is a covalent bond or —CH₂—, the compoundof the present invention is represented by Structural Formula (VI):

R₁-R₆ in Structural Formula (VI) are as described for Structural Formula(I). R₅ and R₆ are the same or different. Y″ is a covalent bond or—CH₂—.

In one example of a compound represented by Structural Formula (VI), R₅and R₆ are both a lower alkyl group (preferably methyl) or a phenylgroup. When R₅ and R₆ are both a lower alkyl group or a phenyl group,then R₁ and R₂ are preferably both phenyl or substituted phenyl and R₃and R₄ are preferably both a lower alkyl group.

The following are more specific examples of compounds of the presentinvention: R₁ and R₂ are both phenyl, R₃ and R₄ are both phenyl, R₅ andR₆ are both methyl, and R₇ and R₈ are both —H; R₁ and R₂ are bothphenyl, R₃ and R₄ are both phenyl, R₅ and R₆ are both n-hexyl, and R₇and R₈ are both —H; R₁ and R₂ are both phenyl, R₃ and R₄ are bothmethyl, R₅ and R₆ are both methyl, and R₇ and R₈ are both —H; R₁ and R₂are both phenyl, R₃ and R₄ are both methyl, R₅ and R₆ are both methyl,and R₇ is —H and R₈ is methyl; R₁ and R₂ are both phenyl, R₃ and R₄ areboth —H, R₅ and R₆ are both phenyl, R₇ is —H, and R₈ is methyl; R₁ andR₂ are both 4-chlorophenyl, R₃ and R₄ are both methyl, R₅ and R₆ areboth methyl, and R₇ and R₈ are both —H.

In Structural Formulas (I)-(VI), R₁ and R₂ are the same or different;and/or R₃ and R₄ are the same or different. Preferably, R₁ and R₂ arethe same, and R₃ and R₄ are the same.

A “straight chained hydrocarbyl group” is an alkylene group, i.e.,—(CH₂)_(x)—, with one or more (preferably one) methylene groupsoptionally replaced with a linkage group. x is a positive integer (e.g.,between 1 and about 10), preferably between 1 and about 6 and morepreferably 1 or 2. A “linkage group” refers to a functional group whichreplaces a methylene in a straight chained hydrocarbyl. Examples ofsuitable linkage groups include a ketone (—C(O)—), alkene, alkyne,phenylene, ether (—O—), thioether (—S—), or amine [—N(R^(a))]—, whereinR_(a) is defined below. A preferred linkage group is —C(R₇R₈)—, whereinR₇ and R₈ are defined above. Suitable substitutents for an alkylenegroup and a hydrocarbaryl group are those which do not substantiallyinterfere with the reactions described herein. R₇ and R₈ are preferredsubstituents for an alkylene or hydrocarbyl group.

An aliphatic group is a straight chained, branched or cyclicnon-aromatic hydrocarbon which is completely saturated or which containsone or more units of unsaturation. Typically, a straight chained orbranched aliphatic group has from 1 to about 20 carbon atoms, preferablyfrom 1 to about 10, and a cyclic aliphatic group has from 3 to about 10carbon atoms, preferably from 3 to about 8. An aliphatic group ispreferably a straight chained or branched alkyl group, e.g, methyl,ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, pentyl,hexyl, pentyl or octyl, or a cycloalkyl group with 3 to about 8 carbonatoms. A C1-C20 straight chained or branched alkyl group or a C3-C8cyclic alkyl group is also referred to as a “lower alkyl” group.

Aromatic groups include carbocyclic aromatic groups such as phenyl,naphthyl, and anthracyl, and heteroaryl groups such as imidazolyl,thienyl, furanyl, pyridyl, pyrimidy, pyranyl, pyrazolyl, pyrroyl,pyrazinyl, thiazole, oxazolyl, and tetrazole.

Aromatic groups also include fused polycyclic aromatic ring systems inwhich a carbocyclic aromatic ring or heteroaryl ring is fused to one ormore other heteroaryl rings. Examples include benzothienyl,benzofuranyl, indolyl, quinolinyl, benzothiazole, benzooxazole,benzimidazole, quinolinyl, isoquinolinyl and isoindolyl.

The term “arylene” refers to an aryl group which is connected to theremainder of the molecule by two other bonds. By way of example, thestructure of a 1,4-phenylene group is shown below:

Substituents for an arylene group are as described below for an arylgroup.

Non-aromatic heterocyclic rings are non-aromatic carbocyclic rings whichinclude one or more heteroatoms such as nitrogen, oxygen or sulfur inthe ring. The ring can be five, six, seven or eight-membered. Examplesinclude tetrahydrofuranyl, tetrahyrothiophenyl, morpholino,thiomorpholino, pyrrolidinyl, piperazinyl, piperidinyl, andthiazolidinyl.

The terms “lower alkoxy”, “lower acyl”, “(lower alkoxy)methyl” and“(lower alkyl)thiomethyl” mean to —O-(lower alkyl), —C(O)—(lower alkyl),—CH₂—O-(lower alkyl) and —CH₂—S-(lower alkyl), respectively. The terms“substituted lower alkoxy” and “substituted lower acyl” mean—O-(substituted lower alkyl) and —C(O)—(substituted lower alkyl),respectively.

Suitable substituents on an aliphatic group, non-aromatic heterocyclicgroup benzylic or aryl group (carbocyclic and heteroaryl) are thosewhich do not substantially interfere with the ability of the disclosedcompounds to enhance the anti-cancer activity of taxol and analogsthereof. A substituent substantially interferes with the ability of adisclosed compound to enhance anti-cancer activity when the enhancementis reduced by more than about 50% in a compound with the substituentcompared with a compound without the substituent. Examples of suitablesubstituents include —OH, halogen (—Br, —Cl, —I and —F), —OR^(a),—O—COR^(a), —COR^(a), —CN, —NO₂, —COOH, —SO₃H, —NH₂, —NHR^(a),—N(R^(a)R^(b)), —COOR^(a), —CHO, —CONH₂, —CONHR^(a), —CON(R^(a)R^(b)),—NHCOR^(a), —NRCOR^(a), —NHCONH₂, —NHCONR^(a)H, —NHCON(R^(a)R^(b)),—NR^(c)CONH₂, —NR^(c)CONR^(a)H, —NR^(c)CON(R^(a)R^(b)), —C(═NH)—NH₂,—C(═NH)—NHR^(a), —C(═NH)—N(R^(a)R^(b)), —C(═NR^(c))—NH₂,—C(═NR^(c))—NHR^(a), —C(═NR^(c))—N(R^(a)R^(b)), —NH—C(═NH)—NH₂,—NH—C(═NH)—NHR ^(a), —NH—C(═NH)—N(R^(a)R^(b)), —NH—C(═NR^(c))—NH₂,—NH—C(═NR^(c))—NHR^(a), —NH—C(═NR^(c))—N(R^(a)R^(b)),—NR^(d)H—C(═NH)—NH₂, —NR^(d)—C(═NH)—NHR^(a),—NR^(d)—C(═NH)—N(R^(a)R^(b)), —NR^(d)—C(═NR^(c))—NH₂,—NR^(d)—C(═NR^(c))—NHR^(a), —NR^(d)—C(═NR^(c))—N(R^(a)R^(b)), —NHNH₂,—NHNHR^(a), —NHR^(a)R^(b), —SO₂NH₂, —SO₂NHR^(a), —SO₂NR^(a)R^(b),—CH═CHR^(a), —CH═CR^(a)R^(b), —CR^(c)═CR^(a)R^(b), —CRH^(c)═CHR^(a),—CR^(c)═CR^(a)R^(b), —CCR^(a), —SH, —SO_(k)R^(a)(k is 0, 1 or 2) and—NH—C(═NH)—NH₂. R^(a)-R^(d) are each independently an aliphatic,substituted aliphatic, benzyl, substituted benzyl, aromatic orsubstituted aromatic group, preferably an alkyl, benzylic or aryl group.In addition, —NR(R^(a)R^(b)), taken together, can also form asubstituted or unsubstituted non-aromatic heterocyclic group. Anon-aromatic heterocyclic group, benzylic group or aryl group can alsohave an aliphatic or substituted aliphatic group as a substituent. Asubstituted aliphatic group can also have a non-aromatic heterocyclicring, a substituted a non-aromatic heterocyclic ring, benzyl,substituted benzyl, aryl or substituted aryl group as a substituent. Asubstituted aliphatic, non-aromatic heterocyclic group, substitutedaryl, or substituted benzyl group can have more than one substituent.

Also included in the present invention are pharmaceutically acceptablesalts of the compounds described herein. The compound of the presentinvention which possess a sufficiently acidic, a sufficiently basic, orboth functional groups, and accordingly can react with any of a numberof inorganic bases, and inorganic and organic acids, to form a salt.Acids commonly employed to form acid addition salts are inorganic acidssuch as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuricacid, phosphoric acid, and the like, and organic acids such asp-toluenesulfonic acid, methanesulfonic acid, oxalic acid,p-bromophenyl-sulfonic acid, carbonic acid, succinic acid, citric acid,benzoic acid, acetic acid, and the like. Examples of such salts includethe sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, acetate, propionate,decanoate, caprylate, acrylate, formate, isobutyrate, caproate,heptanoate, propiolate, oxalate, malonate, succinate, suberate,sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate,phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate,gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate,propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,mandelate, and the like.

Base addition salts include those derived from inorganic bases, such asammonium or alkali or alkaline earth metal hydroxides, carbonates,bicarbonates, and the like. Such bases useful in preparing the salts ofthis invention thus include sodium hydroxide, potassium hydroxide,ammonium hydroxide, potassium carbonate, and the like.

Taxol, also referred to as “Paclitaxel”, is a well-known anti-cancerdrug which acts by inhibiting microtubule formation. Many analogs oftaxol are known, including taxotere, the structure of which is shown inFIG. 4. Taxotere is also referred to as “Docetaxol”. The structure ofother taxol analogs are shown in FIGS. 5-25. These compounds have thebasic taxane skeleton as a common structure feature and have also beenshown to have the ability to arrest cells in the G2-M phases due tostabilized microtubules. Thus, it is apparent from FIGS. 5-25 that awide variety of substituents can decorate the taxane skeleton withoutadversely affecting biological activity. It is also apparent that zero,one or both of the cyclohexane rings of a taxol analog can have a doublebond at the indicated positions. For clarity purposes, the basic taxaneskelton is shown below in Structural Formula (VII):

Double bonds have been omitted from the cyclohexane rings in the taxaneskeleton represented by Structural Formula (VII). It is to be understoodthat the basic taxane skeleton can include zero or one double bond inone or both cyclohexane rings, as indicated in FIGS. 5-25 and StructuralFormulas (VIII) and (IX) below. A number of atoms have also omitted fromStructural Formula (VII) to indicate sites in which structural variationcommonly occurs among taxol analogs. For example, substitution on thetaxane skeleton with simply an oxygen atom indicates that hydroxyl,acyl, alkoxy or other oxygen-bearing substituent is commonly found atthe site. It is to be understood that these and other substitutions onthe taxane skeleton can also be made without losing the ability toenhance and stabilize microtubule formation. Thus, the term “taxolanalog” is defined herein to mean a compound which has the basic taxolskeleton and which promotes disassembly of microtubules.

Typically, the taxol analogs used herein are represented by StructuralFormula (VIII) or (IX):

-   -   R₁₀ is a lower alkyl group, a substituted lower alkyl group, a        phenyl group, a substituted phenyl group, —SR₁₉, —NHR₁₉ or        —OR₁₉.    -   R₁₁ is a lower alkyl group, a substituted lower alkyl group, an        aryl group or a substituted aryl group.    -   R₁₂ is —H, —OH, lower alkyl, substituted lower alkyl, lower        alkoxy, substituted lower alkoxy, —O—C(O)-(lower alkyl),        —O—C(O)—(substituted lower alkyl), —O—CH₂—O—(lower alkyl)        —S—CH₂—O-(lower alkyl).    -   R₁₃ is —H, —CH₃, or, taken together with R₁₄, —CH₂—.    -   R₁₄ is —H, —OH, lower alkoxy, —O—C(O)-(lower alkyl), substituted        lower alkoxy, —O—C(O)—(substituted lower alkyl),        —O—CH₂—O—P(O)(OH)₂, —O—CH₂—O-(lower alkyl), —O—CH₂—S-(lower        alkyl) or, taken together with R₂₀, a double bond.    -   R₁₅ —H, lower acyl, lower alkyl, substituted lower alkyl,        alkoxymethyl, alkthiomethyl, —OC(O)—O(lower alkyl),        —OC(O)—O(substituted lower alkyl), —OC(O)—NH(lower alkyl) or        —OC(O)—NH(substituted lower alkyl).    -   R₁₆ is phenyl or substituted phenyl.    -   R₁₇ is —H, lower acyl, substituted lower acyl, lower alkyl,        substituted, lower alkyl, (lower alkoxy)methyl or (lower        alkyl)thiomethyl.    -   R₁₈ —H, —CH₃ or, taken together with R₁₇ and the carbon atoms to        which R₁₇ and R₁₈ are bonded, a five or six membered a        non-aromatic heterocyclic ring.    -   R₁₉ is a lower alkyl group, a substituted lower alkyl group, a        phenyl group, a substituted phenyl group.    -   R₂₀ is —H or a halogen.    -   R₂₁ is —H, lower alkyl, substituted lower alkyl, lower acyl or        substituted lower acyl.

Preferably, the variables in Structural Formulas (VIII) and (IX) aredefined as follows: R₁₀ is phenyl, tert-butoxy, —S—CH₂—CH—(CH₃)₂,—S—CH(CH₃)₃, —S—(CH₂)₃CH₃, —O—CH(CH₃)₃, —NH—CH(CH₃)₃, —CH═C(CH₃)₂ orpara-chlorophenyl; R₁₁ is phenyl, (CH₃)₂CHCH₂—, -2-furanyl, cyclopropylor para-toluyl; R₁₂ is —H, —OH, CH₃CO— or —(CH₂)₂-N-morpholino; R₁₃ ismethyl, or, R₁₃ and R₁₄, taken together, are —CH₂—;

-   -   R₁₄ is —H, —CH₂SCH₃ or —CH₂—O—P(O)(OH)₂; R₁₅ is CH₃CO—;    -   R₁₆ is phenyl; R₁₇—H, or, R₁₇ and R₁₈, taken together, are        —O—CO—O—;    -   R₁₈ is —H; R₂₀ is —H or —F; and R₂₁ is —H,        —C(O)—CHBr—(CH₂)₁₃—CH₃ or —C(O)—(CH₂)₁₄—CH₃;        —C(O)—CH₂—CH(OH)—COOH, —C(O)—CH₂—O—C(O)—CH₂CH(NH₂)—CONH₂,        —C(O)—CH₂—O——CH₂CH₂OCH₃ or —C(O)—O—C(O)—CH₂CH₃.

A taxol analog can also be bonded to or be pendent from apharmaceutically acceptable polymer, such as a polyacrylamide. Oneexample of a polymer of this type is shown in FIG. 26. The term “taxolanalog”, as it is used herein, includes such polymers.

The disclosed compounds are enhancers of the anti-cancer activity oftaxol and taxol analogs. A compound enhances the anti-cancer activity oftaxol or a taxol analog when the activity of taxol or the taxol analogis greater when administered in combination with the compound than whenadministered alone. The degree of the increase in activity depends uponthe amount of compound administered. The compounds of the presentinvention can therefore be used in combination with taxol or taxolanalogs to treat subjects with cancers. Examples include colon cancer,pancreatic cancer, melanoma, renal cancer, sarcoma, breast cancer,ovarian cancer, lung cancer. stomach cancer, bladder cancer and cervicalcancer.

A “subject” is a mammal, preferably a human, but can also be an animalin need of veterinary treatment, e.g., companion animals (e.g., dogs,cats, and the like), farm animals (e.g., cows, sheep, pigs, horses, andthe like) and laboratory animals (e.g., rats, mice, guinea pigs, and thelike).

In order to achieve an enhancement of the anti-cancer activity of taxoland taxol analogs, an effective amount of a compound of the presentinvention and an effective amount of taxol or analog of taxol areadministered to the subject. With respect to taxol or an analog oftaxol, an “effective amount” is a quantity in which anti-cancer effectsare normally achieved. With respect to a compound of the presentinvention, an “effective amount” is the quantity in which a greateranti-cancer effect is achieved when the compound is co-administered withtaxol or a taxol analog compared with when taxol or the taxol analog isadministered alone. The compound and taxol (or taxol analog) can beco-administered to the subject as part of the same pharmaceuticalcomposition or, alternatively, as separate pharmaceutical compositions.When administered as separate pharmaceutical compositions, the compoundor the present invention and taxol (or taxol analog) can be administeredsimultaneously or at different times, provided that the enhancing effectof the compound is retained.

The amount of compound and taxol (or taxol analog) administered to thesubject will depend on the type and severity of the disease or conditionand on the characteristics of the subject, such as general health, age,sex, body weight and tolerance to drugs. It will also depend on thedegree, severity and type of cancer. The skilled artisan will be able todetermine appropriate dosages depending on these and other factors.Effective dosages for taxol and taxol analog are well known andtypically range from between about 1 mg/mm² per day and about 1000mg/mm² per day, preferably between about 10 mg/mm² per day and about 500mg/mm² per day. Effective amounts of a compound of the present inventiontypically range between about 1 mg/mm² per day and about 10 grams/mm²per day, and preferably between 10 mg/mm² per day and about 5 grams/mm².

The disclosed compounds are administered by any suitable route,including, for example, orally in capsules, suspensions or tablets or byparenteral administration. Parenteral administration can include, forexample, systemic administration, such as by intramuscular, intravenous,subcutaneous, or intraperitoneal injection. The compounds can also beadministered orally (e.g., dietary), topically, by inhalation (e.g.,intrabronchial, intranasal, oral inhalation or intranasal drops), orrectally, depending on the type of cancer to be treated. Oral orparenteral administration are preferred modes of administration.Suitable routes of administration of taxol and taxol analogs are wellknown in the art and include by parenteral administration, as describedabove for the compounds of the present invention. Suitable routes ofadministration for taxol and analogs thereof are well known and includeinter alia parenteral and oral administration.

The disclosed compounds can be administered to the subject inconjunction with an acceptable pharmaceutical carrier as part of apharmaceutical composition for treatment of cancer. Formulation of thecompound to be administered will vary according to the route ofadministration selected (e.g., solution, emulsion, capsule). Suitablepharmaceutical carriers may contain inert ingredients which do notinteract with the compound. Standard pharmaceutical formulationtechniques can be employed, such as those described in Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa. Suitablepharmaceutical carriers for parenteral administration include, forexample, sterile water, physiological saline, bacteriostatic saline(saline containing about 0.9% mg/ml benzyl alcohol), phosphate-bufferedsaline, Hank's solution, Ringer's-lactate and the like. Methods forencapsulating compositions (such as in a coating of hard gelatin orcyclodextrasn) are known in the art (Baker, et al., “Controlled Releaseof Biological Active Agents”, John Wiley and Sons, 1986). Suitableformulations for taxol and taxol analogs are well known in the art.

The disclosed compounds can be prepared according to methods describedin Examples 1-12 and also according to methods described in theco-pending U.S. Provisional Application entitled SYNTHESIS OF TAXOLENHANCERS U.S. Provisional Application No. 60/304,318, filed Jul. 10,2001. The entire teachings of this application are incorporated hereinby reference.

The present invention is illustrated by the following examples, whichare not intended to be limiting in any way.

EXEMPLIFICATION Example 1

Preparation of Thiobenzoic Acid N-methylhydrazide

Thiobenzoic acid N-methylhydrazide were prepared in 88% yield by slightmodification of the prior art (Acta Chem. Scand. 1961, 1087-1096); ¹HNMR (CDCl₃) δ 3.3 (s, 3H), 6.0 (s, 2H), 7.3-7.4 (m, 5H); ESMS calcd(C₈H₁₀N₂S): 166.1; found: 167.1 (M+H)⁺.

Example 2

Preparation of Thiobenzoic acid N-methylhydrazide

Bromobenzene (1.6 g, 10 mmol) was added into 25 ml anhydrous THFsolution containing magnesium powder (0.3 g, 12.5 mmol), and refluxedfor 2 hr. After it was cooled, the clear reaction solution was addedinto carbon disulfide (1 ml, 16.8 mmol) at 0° C., and stirred for 30 minat rt. The resulting mixture was then added into methylhydrazine (1.6ml, 30 mmol) at 0° C., and stirred for another 2 hours. To this solutionwas added water (15 ml) and extracted with EtOAc (30 ml×3). The organicsolution was concentrated to minimum volume, and subjected to silica gelcolumn chromatography (eluant: 1:3−1:1 ethyl acetate: hexanes) to givethiobenzoic acid N¹-methyl hydrazide (0.72 g, total yield: 48%). ¹H NMR(CDCl₃) δ 3.3 (s, 3H), 6.0 (s, 2H), 7.3-7.4 (m, 5H); ESMS calcd(C₈H₁₀N₂S): 166.1; found: 167.1 (M+H)⁺.

Example 3

Preparation of 2,5-Dimethoxythiobenzoic Acid N-methylhydrazine

DCC (4.5 g, 21.8 mmol) was added in one portion to a solution of2,5-dimethoxybenzoic acid (3.6 g, 20 mol), methylhydrazine (1.2 ml, 23mmol) and DMAP (30 mg, cat.) in CH₂Cl₂ (60 ml) cooled in an ice bath.The reaction mixture was stirred overnight at room temperature. Theslurry was cooled at −20° C. for 1 h and filtered. The CH₂Cl₂ solutionwas evaporated and the residue was dried in vacuum. The resulting crudeproduct was dissolved in toluene (50 ml). To this solution was addedLawesson's reagent (5.8 g, 14 mmol). The mixture was refluxed for 40min, cooled to room temperature, and directly subjected to silica gelcolumn chromatography (eluent: 25% to 35% ethyl acetate in hexanes) togive the 2,5-dimethoxythiobenzoic acid N-methylhydrazide (3.7 g, yield:82%) as off-white solid. ¹H NMR (300 MHz, CDCl₃): δ 6.88-6.80 (m, 3H),5.46 (s, 2H), 3.84 (s, 3H), 3.82 (s, 3H), 3.28 (s, 3H).

Example 4

Preparation of N-Malonyl-bis[N′-methyl-N′-(thiobenzoyl)hydrazide]

To a stirred solution of thiobenzoic acid N-methylhydrazide (0.166 g, 10mmol), HOBt H₂O (0.15 g, 11 mmol) and malonic acid (0.052 g, 5 mmol) inDMF (2 mL) was added DCC (0.22 g, 10.7 mmol) at 0° C. The resultantsuspension was stirred at 0° C. for 1 h and at room temperature for 3 h.Precipitated material was filtered off and washed with EtOAc (3×15 mL).Combined filtrate and washings was washed successively with H₂O (2×20mL), 5% citric acid (20 mL), H₂O (20 mL), Saturated NaHCO₃ (20 mL) andbrine (20 mL). After being dried over Na₂SO₄, the solvent was removedunder reduced pressure to afford the crude product as a yellow solid,which was washed with warm EtOAc. 0.16 g (yield 80%) of pure product wasobtained as a yellow powder. R_(f)0.3 (Hexane/EtOAc 1:1 v/v); ¹H NMR(CDCl₃) δ 3.1-3.8 (m, 6H), 3.4 (s, 2H), 7.1-7.45 (m, 10 H), 9.5-10.5 (m,1H) ppm; ESMS calcd (C₁₉H₂₀N₄O₂S₂): 400.1; found: 399.1 (M−H)⁺.Preparation ofN-(2-Methylmalonyl-bis{N′-methyl-N′-[(2,5-dimethoxy)thiobenzoyl]hydrazide]

DCC (4 g, 19 mmol) was added to a solution of 2,5-dimethoxythiobenzoicacid N-methylhydrazide (3.7 g, 16.4 mmol) and 2-methylmalonic acid (2 g,17 mmol) in DMF (20 ml) with stirring at 0° C. The reaction mixture wasstirred for 1 h at room temperature. The slurry was cooled at −20° C.for 1 h and filtered. The filtrate was diluted with EtOAc (300 ml),washed with water (50 ml×3), dried with Na₂SO₄. The EtOAc solution wasconcentrated to minimum volume, and subjected to silica gel columnchromatography (eluent: 1:4 to 2:1, ethyl acetate: hexanes) to give thetitle compound (3.5 g, 80%) as yellow powder. ¹H NMR (CDCl₃) δ10.12-9.14 (2H), 7.12-6.81 (m, 6H), 4.01-3.78 (m, 6H), 3.75-3.22 (m,6H), 2.82-2.62 (m, 1H), 1.12-0.11 (m, 3H); ESMS cacld(C₂₄H₃₀N₄O₆S₂):534.16; found: 535.1 (M+H).

Example 5

Preparation of N-Malonyl-bis[N′-methyl-N′-(thiobenzoyl)hydrazide]

To a solution of thiobenzoic acid N-methylhydrazine (10 g) stirred at 0°C. were added subsequently triethylamine (8.5 mL) and malonyl dichloride(3.05 mL). The reaction mixture was stirred for 10 min, washed withwater (3×50 mL), dried over sodium sulfate and concentrated.Purification by recrystallization from methylene dichloride (35 mL) gavethe product as light yellow crystals (9.0 g, 75%) which was identical tothe product obtained in Example 6.

Example 6

Preparation of N-Malonyl-bis[N′-methyl-N′-(thiobenzoyl)hydrazide]

A stirred solution of thiobenzoic acid N-methylhydrazide (1.66 g, 10mmol) and diphenyl malonate (1.30 g, 5.08 mmol) in dry THF (100 mL) washeated to reflux for 72 h. Volatile components were then removed underreduced pressure. The crude product was purified by columnchromatography on silica gel using a mixture of hexane and EtOAc aseluant (gradient from 4:1 v/v to 1:1 v/v). 1.07 g (51% yield) of pureproduct N-malonyl-bis[N′-methyl-N′-(thiobenzoyl)hydrazide] was obtainedas a yellow powder. Physical property was identical to that obtained inExample 5.

Example 7

A slurry of thiobenzoic acid N-methylhydrazide (1.0 g, 6 mmol),mono-tert-butyl malonate (1.0 mL, 6 mmol), HOBt.H₂O (0.98 g, 7.2 mmol),and DCC (1.34 g, 6.5 mmol) in DMF (5 mL) was stirred at 0° C. for 3 hand then at room temperature for 3 h. Precipitated material was filteredoff and washed with EtOAc (3×20 mL). Combined filtrate and washings waswashed successively with H₂O (2×20 mL), 5% citric acid (20 mL), H₂O (20mL), Saturated NaHCO₃ (20 mL) and brine (20 mL). After being dried overNa₂SO₄, the solvent was removed under reduced pressure to afford thecrude product as a solid, which was washed with Et₂O. 0.94 g (yield 51%)of pure product N′-Methyl-N′-thiobenzoyl-hydrazinocarbonyl)-acetic acidtert-butyl ester was obtained as a yellow powder. ¹H NMR (CDCl₃) δ1.6-1.7 (ds, 9H), 3.1-4.1 (m, 5H), 7.3-7.7 (m, 5H), 9.7-10.3 (ds,1H)ppm; ESMS calcd (C₁₅H₂₀N₂O₃S): 308; found: 307 (M−H)⁺.

A solution of N′-methyl-N′-thiobenzoyl-hydrazinocarbonyl)-acetic acidtert-butyl ester (0.19 g, 0.6 mmol) and TFA (0.12 mL, 1.6 mmol) in dryDCM (10 mL) was stirred at 10° C.-15° C. for 12 h (reaction wasmonitored by TLC). Volatile components were removed under reducedpressure (bath temperature below 5° C.). After being dried in vacuo, DMF(3 mL) was added followed by the addition of DCC (0.13 g, 0.6 mmol),HOBt H₂O (93 mg, 0.7 mmol) and thio-2,5-dimethoxybenzoic acidN-methylhydrazide (0.13 g, 0.57 mmol). The resultant solution wasstirred at 0° C. for half an hour and then at room temperature for 3 h.Precipitated material was filtered off and washed with EtOAc (3×10 mL).Combined filtrate and washings was washed successively with H₂O (2×10mL), 5% citric acid (10 mL), H₂O (10 mL), Saturated NaHCO₃ (20 mL) andbrine (20 mL). After being dried over Na₂SO₄, the solvent was removedunder reduced pressure to afford the crude product as an oil, which waspurified by SGC (4:1 hexane/EA to 2:1 EtOAc/Hexane). 0.14 g (yield 53%)of pure product was obtained as a yellow powder. ¹H NMR (CDCl₃) δ3.1-3.9 (m, 18H), 6.7-7.4 (m, 9H)ppm; ESMS calcd (C₂₁H₂₄N₄O₄S2): 460.1;found: 461.1 (M+H)⁺.

Example 8

Preparation of N-N-malonyl-bis[N′-phenyl-N′-(thiobenzoyl)hydrazide]

A mixture of phenylhydrazine (30 mL) and ethyl malonate (in xylene (150mL) was heated to reflux overnight. The reaction was cooled to roomtemperature. The precipitates were collected via filtration and washedwith ethanol to give N-malonyl-bis(N′-phenylhydrazide) as a white solid(14 g). The hydrazide (3.4 g) was suspended in benzoic anhydride (50 g)with warming. To it was added dropwise perchloric acid (57% in water, 3mL). The reaction mixture turned to clear solution initially and thenquickly solidified. After standing at room temperature for 1 h, ether(50 mL) was added. The resulting slurry was filtered and washed withether (2×00 mL) to give the perchlorate salts as a white solid (5.7 g).The salts were taken into acetone and added as a slurry over 5 min toNa₂S (0.6 M in water, 90 mL) stirred at room temperature. After 30 min,the reaction was acidified with HCl(c) to afford a yellow slurry. Thesolid was collected via filtration and washed with water (20 mL) andether (2×25 mL) to giveN-malonyl-bis[N′-phenyl-N′-(thiobenzoyl)hydrazide] as an off-white solid(3.6 g). ¹H NMR (CDCl₃): δ7.2 (m, 20H); 3.5 (br s, 2H). MS calcd forC₂₉H₂₄N₄O₂S₂: 524.13: Found: 525.1 (M+H).

Example 9

Preparation ofN-Malonyl-bis[N-methyl-N′-phenyl-N′-(thiobenzoyl)hydrazide]

To a stirred solution ofN-malonyl-bis[N′-phenyl-N′-(thiobenzoyl)hydrazide] (180 mg, 0.34 mmol),MeOH (22 uL) and triphenylphosphine (200 mg, 0.64 mmol) in dry THF(10mL) was added a solution of DEAD (0.12 mL) in THF (3 mL) dropwise. Theresultant orange solution was stirred at room temperature for 12 h.After removal of the volatile components, the crude product was purifiedby SGC (3:1 Hexane/EtOAc) to afford 98 mg (52% yield) of the titlecompound as syrup. ¹H NMR (CDCl₃) δ 3.3-4.5 (m, 8H), 7.1-7.8 (m, 20H)ppm; ESMS calcd (C₃₁H₂₈N₄O₂S₂): 552; found: 551 (M−H)⁺.

Example 10

A stirred mixture of N-malonyl-bis[N′-phenyl-N′-(thioacetyl)hydrazide)starting material (0.1 g, 0.25 mmol) and Lawesson's reagent (0.15 g,0.37 mmol) in dry benzene (20 mL) was heated to reflux for 1 h. Afterbeing cooled to room temperature, the mixture was filtered through alayer of silica gel, washed with THF (2×15 mL). The filtrate andwashings were combined and concentrated under reduced pressure. Flushcolumn chromatography on silica gel (hexane to 4:1 hexane/EtOAc to 2:1hexane/EtOAc) affordedN-bisthiomalonyl-bis[N′-phenyl-N′-(thioacetyl)hydrazide as a clear syrup(16 mg, 15%). ¹H NMR (CDCl₃) δ 3.80-3.95 (m, 8H), 7.02-7.30 9 m, 10 H).ESMS calcd (C₁₉H₂₀N₄S₄): 432.06; found: 433.0 (M+H)⁺.

Example 10A

Cyclopropyl bromide (4.8 g, 40 mmol) was added into 50 ml anhydrous THFsolution containing magnesium powder (1.1 g, 45 mmol), stirred for 30min, and refluxed for another 30 min. After it was cooled, the clearreaction solution was added into carbon disulfide (4 ml, 67 mmol) at 0°C., and stirred for 30 min at rt. The resulting mixture was then addedinto methylhydrazine (8 ml, 150 mmol) at 0° C., and stirred for another2 hours. To this solution was added water (40 ml) and extracted withEtOAc (60 ml×3). The organic solution was concentrated to minimumvolume, and subjected to silica gel column chromatography (1:1 ethylacetate: hexanes; ethyl acetate) to give thiocyclopropyl carboxylic acidN¹-methyl hydrazide (2.8 g, 55%). ¹H NMR (300 MHz, CDCl₃): δ 5.21 (br.,2H), 3.62 (s, 3H), 1.91 (m, 1H), 1.25 (m, 2H), 0.98 (m, 2H). ESMS cacld(C₅H₁₀N₂S): 130.1; found: 131.1 (M+H)⁺. To the hydrazide EtOAc solution(2.8 g, 22 mmol, 40 ml) containing TEA (2.2 g, 22 mmol) was addedmalonyl chloride EtOAc solution (1.6 g, 11 mmol, 4 ml) at 0° C., and thereaction mixture was stirred at rt for 20 min. 20 ml water was added toquench the reaction, and the EtOAc layer was continuously washed twicewith water (20 ml×2). The EtOAc solution was concentrated to minimumvolume, and subjected to silica gel column chromatography (eluant:1:1-1:2 hexanes: ethyl acetate) to give SBR-11-5685 (2.1 g, yield: 60%).(2.1 g, yield: 60%). ¹H NMR (300 MHz, CDCl₃): δ 10.01-8.95 (m, 2H),3.78-3.41 (m, 6H), 2.34-0.82 (m, 10H). ESMS cacld(C₁₃H₂₀N₄O₂S₂): 328.1;found: 327 (M−H)⁺.

Example 10B

The compound shown above was prepared by the procedure provided inExample 10B. ¹H NMR (300 MHz, CDCl₃): δ 9.79(br, 2H), 3.79-3.41 (m, 6H),1.60-0.75(m, 18H). ESMS cacld(C₁₅H₂₄N₄O₂S₂): 356.13; found: 355 (M−H)⁺.

Example 11

The compounds shown below were prepared by the procedures describedabove. Analytical data is provided for these compounds.

-   -   ¹H NMR (CDCl₃) δ 3.1-3.8 (m, 6H), 3.4 (s, 2H), 7.1-7.45 (m, 10        H), 9.5-10.5 (m, 1H) ppm; ESMS calcd (C₁₉H₂₀N₄O₂S₂): 400.1;        found: 399.1 (M−H)⁺.    -   ¹H NMR (CDCl₃) δ 1.0-1.35 (m, 6H), 3.0-4.3 (m, 6H), 7.05-7.40        (m, 10H), 9.1-10.1 (m, 2H); ESMS cacld (C₂H₂₄N₄O₂S₂): 428.8;        found: 427 (M−H)⁺. Anal Calc For C₂₁H₂₄N₄O₂S₂ (428.13) C, 58.85;        H, 5.64; N, 13.07; S, 14.96. Found: C, 58.73; H, 5.62; N, 12.97;        S 14.96.    -   ¹H NMR (CDCl₃) δ 0.7-1.0 (m, 6H), 1.4-1.9 (m, 4H), 3.1-4.2 (m,        6H), 7.1-7.4 (m, 10H), 8.9-10.2 (m, 2H) ppm; ESMS        (C₂₃H₂₈N₄O₂S₂): 456.1; found: 455.1 (M−H)⁺.    -   mp 141-143° C.; ¹H NMR (CDCl₃) δ 0.6-1.05 (m, 6H), 1.1-1.9 (m,        8H), 3.0-4.2 (m, 6H), 7.0-7.35 (m, 10H), 8.9-11 (ms, 2H). ESMS        (C₂₅H₃₂N₄O₂S₂): 484.2; found 483.1 (M−H)⁺. Anal Calc For        C₂₅H₃₂N₄O₂S₂ (484.2) C, 61.95; H, 6.65; N, 11.56; S, 13.23.        Found: C, 61.98; H, 6.52; N, 11.26; S, 13.16.    -   ¹H NMR (DMSO-d₆) δ 0.4-0.9 (dd, 3H, J=7), 2.7 (q, 1H), 3.1-3.6        (m, 6H), 7.1-7.5 (m, 10H), 10.9 (br, 2H)ppm; ESMS        (C₂₀H₂₂N₄O₂S₂): 414; found: 413 (M−H)⁺.    -   ¹H NMR (CDCl₃) δ 0.5 (t, 3H, J=7), 1.1-1.6 (m, 2H), 2.7 (t, 1H,        J=7), 3.1-3.3 (m, 6H), 7.0-7.3 (m, 10H), 10.25 (s, 2H)ppm; MS        (C₂₁H₂₄N₄O₂S₂): 428.1; found: 427.1 (M−H)⁺.    -   ¹H NMR (CDCl₃) δ 0.5 (d, 6H, J=7), 0.9-1.2 (m, 1H), 3.0-41 (m,        7H), 7.1-7.4 (m, 10H), 10.3 (s, 2H)ppm; ESMS (C₂₂H₂₆N₄O₂S₂):        442.1; found: 441.1 (M−H)⁺.    -   ¹H NMR (CDCl₃) δ 0.4-1.3 (m, 5H), 1.5-1.8 (m, 2H), 3.0-3.7 (m,        6H), 7.1-7.5 (m, 10H), 11 (s, 2H) ppm; ESMS (C₂₃H₂₈N₄O₂S₂):        456.1; found: 455.1 (M−H)⁺.    -   ¹H NMR (CDCl₃) δ 2.1 (d, 2H, J=7), 2.9 (t, 1H, J=7), 3.1-3.5 (m,        6H), 6.8-7.4 (m, 15H), 11 (s, 2H)ppm; ESMS (C₂₆H₂₆N₄O₂S₂):        490.1; found: 489.1 (M−H)⁺.    -   ¹H NMR (CDCl₃) δ 0.4 (d, 3H, J=7), 1.0-1.4 (m, 6H), 2.75 (q,        1H), 3.0-4.3 (m, 4H), 7.1-7.4 (m, 10H), 10.6 (s, 2H); ESMS Calc        For (C₂₂H₂₆N₄O₂S₂): 442.1; found: 441.1 (M−H)⁺; Anal Calc For        C₂₂H₂₆N₄O₂S₂ (442.15) C, 59.70; H, 5.92; N, 12.66; S, 14.49.        Found: C, 59.64; H, 5.92; N, 12.59; S, 14.47.    -   ¹H NMR (DMSO-d₆) δ 3.20 (br, 2H), 7.1-7.6 (m, 20 H), 11.5 (s,        2H) ppm; ESMS calcd (C₂₉H₂₄N₄O₂S₂): 524.1; found: 523.1 (M−H)⁺.    -   ¹H NMR (CDCl₃) δ 3.0-4.3 (m, 14H), 6.6-7.5 (m, 8H), 10.4 (s, 2H)        ppm; ESMS calcd (C₂₁H₂₄N₄O₂S₂): 460.2; found: 461.2 (M+H)⁺.    -   ¹H NMR (CDCl₃) δ 2.65-3.60 (m, 8H), 7.2-7.4 (m, 8H), 11.1 (br,        2H); ESMS calcd (C₁₉H₁₈Cl₂N₄O₂S₂): 468.0; found: 467.9 (M−H)⁺.    -   ¹H NMR (CDCl₃) δ 0.4 (d, 3H, J=7), 2.7 (q, 1H, J=7), 3.0-3.8 (m,        6H), 7.2-8.2 (m, 8H), 10.5-10.7 (ms, 2H) ppm; ESMS calcd        (C₂₀H₂₀Cl₂N₄O₂S₂): 482.0; found: 481.0 (M−H)⁺.    -   ¹H NMR (CDCl₃) δ 2.9-3.8 (m, 6H), 7.3-7.7 (m, 4H), 8.0-8.3 (m,        4H), 0.9 (s, 2H); ESMS calcd (C₁₀H₁₈N₆O₆S₂): 490.0; found: 489.0        (M−H)⁺.    -   ¹H NMR (CDCl₃) δ 3.1-3.9 (m, 14H), 6.7-7.8 (m, 8H), 9.0-10 (m,        2H) ppm; ESMS calcd (C₂₁H₂₄N₄O₄S₂): 460.1; found: 459.1 (M−H)⁺.    -   (SBR-11-5032): ¹H NMR (CDCl₃) δ 3.0-3.9 (m, 14H), 6.7-7.3 (m,        8H), 9.0-10 (m, 2H) ppm; ESMS calcd (C₂₁H₂₄N₄O₄S₂): 460.1;        found: 459.1 (M−H)⁺.    -   ¹H NMR (acetone-d₆) δ 3.5 (s, 2H), 6.45 (d, 2H, J=5), 6.9 (d,        2H, J=5), 7.2-7.6 (m, 12H), 10.6 (s, 2H) ppm; ESMS calcd        (C₂₅H₂₀N₄O₄S₂): 504.1; found: 503.1 (M−H)⁺.    -   ¹H NMR (DMSO-d6) δ 2.60 (s, 6H), 3.05 (s, 6H), 3.40 (s, 2H),        7.15-7.50 (m, 8H)ppm; ESMS calcd (C₂₇H₂₄Cl₂N₆O₄S₂): 630.1;        found: 629.1 (M−H)⁺.    -   ¹H NMR (CDCl₃) δ 10.06-8.82 (2H), 7.16-6.81 (m, 6H), 4.01-3.8 1        (m, 6H), 3.78-3.11 (m, 6H), 2.81-2.58 (m, 2H): ESMS cacld        (C₂₃H₂₈N₄O₆S₂): 520.15; found: 521 (M+H).    -   ¹H NMR (CDCl₃) δ 10.38-9.01 (2H), 7.12-6.82 (m, 6H), 3.92-3.78        (m, 12H), 3.75-3.06 (m, 6H), 2.61-2.51 (m, 2H); ESMS cacld        (C₂₃H₂₈N₄O₆S₂): 520.15; found: 521 (M+H).    -   ¹H NMR (CDCl₃) δ 9.45-8.63 (2H), 7.18-6.81 (m, 6H), 4.01-3.80        (m, 6H), 3.78-3.24 (m, 6H), 2.62-2.50 (m, 1H), 1.74-0.11 (m,        3H); ESMS cacld (C₂₄H₃₀N₄O₆S₂): 534.16; found: 535 (M+H).    -   ¹H NMR (CDCl₃) δ 10.19-8.61 (2H), 7.26-6.52 (m, 6H), 3.81-3.08        (m, 8H), 3.01-2.88 (m, 12H). ESMS cacld (C₂₃H₃₀N₆O₂S₂): 486.19;        found: 487 (M+H).    -   ¹H NMR (CDCl₃) δ 9.92-8.80 (2H), 7.41-6.72 (m, 6H), 4.01-3.81        (m, 6H), 3.80-3.15 (m, 6H), 2.76-2.42 (m, 2H); ESMS cacld        (C₂₁H₂₂Cl₂N₄O₄S₂):528.05; found: 529(M+H).    -   ¹H NMR (CDCl₃) δ 10.21-9.02(2H), 7.60-6.81 (m, 6H), 4.14-3.88        (m, 6H), 3.87-3.18 (m, 6H), 2.84-2.65 (m, 1H), 1.10-0.16 (m,        3H); ESMS cacld (C₂₂H₂₄Cl₂N₄O₄S₂): 542.06; found: 543 (M+H).    -   ¹H NMR (CDCl₃) δ 10.02-9.20 (2H), 7.63-7.01 (m, 6H), 4.21-3.22        (m, 6H), 1.88-1.36 (m, 2H); ESMS cacld (C₁₉H₁₆F₄N₄O₂S₂): 472.07;        found: 473 (M+H).    -   ¹H NMR (CDCl₃) δ 7.93-7.61 (2H), 7.40-6.92 (m, 6H), 3.98-3.41        (m, 6H), 2.19-0.93 (m, 4H); ESMS cacld (C₂₀H₁₈F₄N₄O₂S₂): 486.08;        found: 487 (M+H).    -   ¹H NMR (CDCl₃) δ 10.12-9.21 (2H), 7.67-7.23 (m, 6H), 3.94-3.22        (m, 6H), 2.01-1.21 (m, 2H); ESMS cacld (C₁₉H₁₆Cl₄N₄O₂S₂):        535.95; found: 537(M+H).    -   ¹H NMR (CDCl₃) δ 7.78-7.23 (2H), 4.56-3.10 (m, 6H), 2.34-1.12        (m, 4H), ESMS cacld (C₂₀H₁₈Cl₄N₄O₂S₂): 549.96; found: 551 (M+H).    -   ¹H NMR (CDCl₃) δ 9.92-9.01 (2H), 7.38-7.15 (m, 3H), 6.66-6.51        (m, 3H), 3.98-3.75 (m, 12H), 3.72-3.21(m, 6H), 2.01-0.42 (m,        4H); ESMS cacld (C₂₄H₃₀N₄O₆S₂): 534.16; found; 535 (M+H).    -   ¹H NMR (CDCl₃) δ 10.51-9.82 (2H), 7.42-6.80 (m, 6H), 3.92-3.04        (m, 6H), 2.60-1.21 (m, 14H); ESMS cacld (C₂₃H₂₈N₄O₂S₂): 456.17;        found: 457(M+H).    -   ¹H NMR (CDCl₃) δ 10.51-8.82 (2H), 7.11-6.89 (m, 6H), 3.81-3.02        (m, 6H), 2.40-1.02 (m, 16H); ESMS cacld (C₂₄H₃₀N₄O₂S₂): 470.18;        found: 471(M+H).    -   ¹H NMR (CDCl₃) δ 9.86-8.42 (2H), 7.01-6.6 (m, 6H), 4.18-3.51 (m,        16H), 3.22-2.26 (2H), 1.40-1.04 (m, 6H); ESMS cacld        (C₂₅H₃₂N₄O₆S₂):548.18; found: 547 (M−H).    -   ¹H NMR (CDCl₃) δ 9.99-8.41 (2H), 7.01-6.68 (m, 6H), 4.18-3.56        (m, 16H), 1.40-0.02 (m, 10H); ESMS cacld (C₂₆H₃₄N₄O₆S₂): 562.19;        found: 561(M−H).    -   ¹H NMR (CDCl₃) δ 10.12-8.82 (2H), 7.03-6.62 (m, 6H), 4.21-3.87        (m, 8H), 3.84-3.01 (m, 6H), 2.71-2.42 (m, 2H), 1.56-1.21 (m,        12H); ESMS cacld (C₂₇H₃₆N₄O₆S₂): 576.21; found: 577(M+H).    -   ¹H NMR (CDCl₃) δ 9.81-8.79 (2H), 7.01-6.64 (m, 6H), 4.21-3.81        (m, 8H), 3.80-3.22 (m, 6H), 1.54-1.20 (m, 13H), 1.01-0.16 (m,        3H); ESMS cacld (C₂₈H₃₈N₄O₆S₂): 590.22; found: 591 (M+H).    -   ¹H NMR (DMSO-d₆): δ 8.25 (d, J=8.1 Hz, 4H), 7.50 (d, J=8.1 Hz,        4H), 3.7-3.3 (m, 8H); ESMS cacld for C₁₉H₁₈N₆O₆S₂: 490.1; Found:        489.0 (M−H).    -   ¹H NMR (CDCl₃): δ 10.25 (m, 2H), 7.7-7.4 (m, 8H), 3.7 (m, 2H),        3.35 (m, 6H); ESMS cacld for C₂₁H₁₈N₆O₂S₂: 450.1; Found: 449.0        (M−H).    -   ¹H NMR (CDCl₃): δ 8.2 (s, 2H), 7.7-7.5 (m, 4H), 3.7-3.4 (m, 8H),        2.9-2.8 (m, 6H); ESMS cacld for C₁₉H₂₂N₆O₂S₂: 430.1; Found:        431.1 (M+H).    -   ¹H NMR (CDCl₃): δ 10.0-9.2 (m, 2H), 7.9-7.45 (m, 8H), 4.0-3.4        (m, 8H); ESMS cacld for C₂₁H₁₈N₆O₂S₂: 450.1; Found: 451.0 (M+H).    -   ¹H NMR (CDCl₃): δ 10.1-9.4 (2H), 7.5-7.2 (m, 8H), 3.9-3.3 (m,        8H); ESMS cacld for C₁₉H₁₈F₂N₄O₂S₂: 436.1; Found: 437.1 (M+H).    -   ¹H NMR (CDCl₃): δ 3.3 (s, 2H), 3.6 (s, 6H), 5.25 (s, 4H),        7.05-7.3 (m, 16H), 7.6 (s, 2H), 7.9 (d, 2H, J=6), 10.56 (s,        2H)ppm; ESMS calcd (C₃₇H₃₄N₆O₂S₂): 658.2; found: 659.2 (M+H).    -   ¹H NMR (DMSO) δ 11.98 (2H), 7.44-7.12 (m, 10H), 3.69-3.14 (s,        6H). ESMS cacld (C₁₈H₁₈N₄O₂S₂): 386.09: found: 387.1 (M+H).    -   ¹H NMR (CHCl₃) δ 9.48-8.55 (2H), 7.56-7.20 (m, 10H), 3.80-3.31        (m, 6H), 2.88-2.22 (m, 4H). ESMS cacld (C₂₀H₂₂N₄O₂S₂): 414.12;        found: 415.1 (M+H).    -   ¹H NMR (300 MHz, CDCl₃) δ 10.21-9.91 (m, 2H), 8.06-7.32 (m,        14H), 3.91-3.56 (m, 6H). ESMS cacld (C₂₄H₂₂N₄O₂S₂): 462.12;        found: 463 (M+H).    -   ¹H NMR (300 MHz, DMSO-d₆) δ 11.60-11.40 (m, 2H), 7.48-6.46 (m,        12H), 3.64-3.3.30 (m, 6H). ESMS cacld (C₂₀H₂₀N₄O₂S₂): 412.10;        found: 413 (M+H).    -   ¹H NMR (300 MHz, CDCl₃) δ 7.58-7.20 (m, 12H), 3.68-3.20 (m, 6H).        ESMS cacld (C₂₀H₂₀N₄O₂S₂): 412.10; found: 413 (M+H).    -   ¹H NMR (300 MHz, CDCl₃): δ 9.65-8.70 (2H), 8.01-7.21 (m, 14H),        3.84-3.40(m, 6H). ESMS cacld (C₂₄H₂₂N₄O₂S₂): 462.12: found: 463        (M+H).    -   ¹H NMR (CDCl₃): δ 7.2 (m, 18H); 3.5 (br s, 2H); 2.4 (br s, 6H).        MS calcd for C₃₁H₂₈N₄O₂S₂: 552.2: Found: 553.2 (M+H).    -   ¹H NMR (CDCl₃): δ 7.5 (br s, 18H), 3.4 (br s, 2H), 2.45 (s, 6H).        ESMS cacld for C₃₃H₂₈N₄O₆S₂: 640.1; Found 641.1 (M+H).    -   ¹H NMR (CDCl₃—D₂O): δ 7.45-7.15 (m, 20 H), 1.6 (br s, 6H). ESMS        cacld for C₃₁H₂₈N₄O₂S₂: 552.2; Found: 553.2 (M+H).    -   ¹H NMR (DMSO-d₆): δ 11.3 (s, 2H), 7.75 (d, J=6.0 Hz, 2H),        7.5-7.4 (m, 12 H); 6.9 (m, 2H); ESMS cacld for C₂₇H₂₄N₄O₂S₄:        564.1; Found: 565.2 (M+H).    -   ¹H NMR (300 MHz, CDCl₃): δ 10.18-8.60 (m, 2H), 7.26-6.46 (m,        8H), 3.80-3.02(m, 6H), 3.00-2.80 (m, 12H). 1.78-1.56 (m, 2H).        ESMS cacld(C₂₃H₃₀N₄O₂S₂): 486.19; found 487 (M+H).    -   ¹H NMR (300 MHz, DMSO): δ 10.90-10.81 (m, 2H), 7.50-7.21 (m,        10H), 3.78-3.36 (m, 6H), 2.64-0.50 (m, 10H). ESMS        cacld(C₂₀H₂₈N₄O₂S₂): 456.17; found: 457 (M+H).    -   ¹H NMR (300 MHz, CDCl₃): δ 10.00-9.71 (m, 2H), 7.72-7.21 (m,        8H), 3.80-3.26(m, 6H). ESMS cacld(C₂₀H₁₆N₆O₂S₂):436.08; found:        437 (M+H).    -   ¹H NMR (300 MHz, CDCl₃): δ 10.60-9.41 (m, 2H), 7.15-6.23 (m,        6H), 3.89-3.28(m, 6H), 3.76 (S, 12H). ESMS        cacld(C₂₂H₂₈N₄O₆S₂):506.13; found:507 (M+H).    -   ¹H NMR (300 MHz, DMSO): δ 7.40-7.12 (m, 10H), 3.70-2.80 (m, 6H),        1.84-0.72 (m, 16H). ESMS cacld(C₂₆H₃₄N₄O₂S₂):498.21; found: 499        (M+H).    -   ¹H NMR (300 MHz, CDCl₃): δ 10.42-9.53 (m, 2H), 7.55-6.87 (m,        8H), 3.99-3.28 (m, 6H), ESMS cacld(C₁₈H₁₀N₄F₂O₂S₂): 422.07;        found: 423 (M+H).    -   ¹H NMR (300 MHz, DMSO): δ 12.08 (br. 2H), 8.27-7.24 (m, 8H),        3.70-3.15 (m, 6H). ESMS cacld(C₁₈H₁₆N₆O₆S₂):476.06; found: 477        (M+H).    -   ¹H NMR (300 MHz, CDCl₃): δ 10.12-9.83 (m, 2H), 7.15-6.63 (m,        6H), 3.99-2.91 (m, 6H), ESMS cacld(C₂₂H₂₆N₄O₆S₂):506.13; found:        507 (M+H).    -   ¹H NMR (300 MHz, DMSO): δ 11.12-10.54 (m, 2H), 8.27-7.18 (m,        10H), 4.26-3.72 (m, 2H), 3.37-3.18 (m, 2H). ESMS        cacld(C₁₇H₁₆N₄O₂S₂):372.07; found: 371 (M−H).    -   ¹H NMR (300 MHz, DMSO): δ 11.52 (br, 2H), 7.95-7.33 (m, 10H),        3.42-3.22 (m, 6H), 2.48 (m, 2H). ESMS        cacld(C₂₃H₂₀N₄O₂S₄):512.05; found: 513 (M+H).    -   ¹H NMR (300 MHz, CDCl₃): δ 7.81-7.28 (m, 8H), 3.82 (s, 6H). ESMS        cacld(C₂₂H₁₈N₄O₂S₄):498.03; found: 499 (M+H).    -   ¹H NMR (300 MHz, CDCl₃): δ 10.02-9.11 (m, 2H), 8.16-7.28 (m,        8H), 3.99-3.08 (m, 6H), 2.90-1.20 (m, 2H). ESMS        cacld(C₂₃H₂₄N₄O₆S₂):516.11; found: 517 (M+H).    -   ¹H NMR (300 MHz, DMSO): δ 7.99 (m, 8H), 8.16-7.28 (m, 8H),        3.80-3.14 (m, 6H), 1.80-1.21 (m, 2H). ESMS        cacld(C₂₁H₂₀N₄O₆S₂):488.08; found: 487 (M−H).    -   ¹H NMR (300 MHz, CDCl₃): δ 10.82-10.55 (m, 2H), 7.91-7.29 (m,        10H), 3.64−3.11 (m, 6H), 1.90-1.40 (m, 2H). ESMS        cacld(C₁₉H₂₀N₄O₂S₂):400.19; found: 399 (M−H).

Example 12

Compound (1) Enhances the Anti-Cancer Activity of Paclitaxel in vivo

General Procedure of in vivo Anti-Tumor Study

The in vivo anti-cancer enhancing effect of novel compounds was assessedin tumor bearing mice using the tumor growth inhibition assay. Tumorcells were implanted by injection of a tumor cell suspensionsubcutaneously in the flank of a mouse. Treatment of the tumor with anexperimental compound and Paclitaxel begun after the tumor had beenestablished (volume was about 100 mm³). Animal then begun a multipleinjection schedule where the compound and Paclitaxel were given by IVroute of administration. Tumors were measured two times a week. Duringthe course of this assay, animals were monitored daily for signs oftoxicity including body weight loss.

Procedure

A supplemented media was prepared from 50% DMEM/Dulbecco Modified EagleMedium (High Glucose), 5.0% RPMI 1640, 10% FBS/Fetal Bovine Serum(Hybridoma Tested; Sterile Filtered), 1% L-Glutamine, 1%Penicillin-Streptomycin, 1% MEM Sodium Pyruvate and 1% MEM Non-EssentialAmino Acids. FBS was obtained from Sigma Chemical Co. and otheringredients were obtained from Invitrogen Life Technologies, USA). Thesupplemental media was warmed to 37° C. and 50 ml of media was added toa 175 cm² tissue culture flask.

The cells used in the assay were MDA-435 Human Breast Carcinoma from theAmerican Type Culture Collection. 1 vial of MDA-435 cells from theliquid nitrogen frozen cell stock was removed. The frozen vial of cellswas immediately placed into a 37° C. water bath and gently swirled untilthawed. The freeze-vial was wiped with 70% ethanol and cells wereimmediately pipetted into the 175 cm² tissue culture flask containingsupplemented media. The cells were incubated overnight and the media wasremoved and replaced with fresh supplemented media the next day. Theflask was incubated until flask became about 90% confluent. This tookanywhere from 5-7 days.

The flask was washed with 10 ml of sterile room temperature phosphatebuffered saline (PBS). The cells were trypsinized by adding 5 ml ofwarmed Trypsin-EDTA (Invitrogen) to the flask of cells. The cells werethen incubated for 2-3 minutes at 37° C. until cells begun to detachfrom the surface of the flask. An equal volume of supplemented media (5ml) was added to the flask. All the cells were collected into 50 mltube, and centrifuged at 1000 RPM for 5 minutes at 20° C. Thesupernatant was aspirated and the cell pellet was resuspended in 10 mlof supplemented media and the cells were counted. 1-3 millioncells/flask were seeded into 5-7 tissue culture flasks (175 cm²). Eachflask contained 50 ml of supplemented media. The flasks were incubateduntil about 90% confluent. The passaging of the cells was repeated untilenough cells have been grown for tumor implantation.

The above procedure for trypsinizing and centrifuging the cells werefollowed. The supernatant was aspirated and the cell pellet wasresuspended in 10 ml of sterile PBS and the cells were counted. Thecells were centrifuged and then resuspended with appropriate volume ofsterile PBS for injection of correct number of cells needed for tumorimplantation. In the case of MDA-435, 100 million cells were suspendedwith 2.0 ml of sterile PBS to a final concentration of 50 millioncells/ml in order to inject 5 million cells in 0.1 ml/mouse.

Mice (CD-1 nu/nu) were obtained from Charles River Laboratories:nomenclature: Crl:CD-1-nuBR, Age: 6-8 weeks. The mice were allowed toacclimate for 1 week prior to their being used in an experimentalprocedure.

Implantation of the MDA-435 tumor cell suspension took place into thecorpus adiposum of the female CD-1 nu/nu mouse. This fat body is locatedin the ventral abdominal viscera of the mouse. Tumor cells wereimplanted subsutaneously into the fat body located in the right quadrantof the abdomen at the juncture of the os coxae (pelvic bone) and the osfemoris (femur). 5 million MDA-435 cells in 0.1 ml of sterile PBS wereinjected using 27 G (½ inch) needle. MDA-435 tumors developed 2-3 weeksafter implantation.

Compound stock solutions were prepared by dissolving the compound incell-culture-grade DMSO (dimethyl sulfoxide) at the desiredconcentration. This stock solution in DMSO was sonicated in anultrasonic water bath until all the powder dissolved.

The Formulation Solvent was prepared as follows: 20% of Cremophore RH40(Polyoxyl 40 Hydrogenated Castor Oil obtained from BASF corp.) in waterwas prepared by first heating 100% Cremophore RH40 in a water bath at50-60° C. until it liquefied and became clear. 10 ml of the 100%Cremophore RH40 aliquoted into a conical centrifuge tube containing 40ml of sterile water (1:5 dilution of Cremophore RH40). The 20%Cremophore RH40 solution was reheated until it became clear again, andmixed by inverting the tube several times. This 20% Cremophore RH40solution was stored at room temperature, and was kept for up to 3months.

Preparation of Dosing Solution for Compound Administration: The compoundstock solution was diluted 1:10 with 20% Cremophore RH40:1) 2.0 ml of 10mg/ml dosing solution of Compound (1) was prepared by diluting 100 mg/mlCompound Stock solution with 1.8 ml of 20% Cremophore RH40 watersolution; and 2) a dosoing solution comprising 2.0 ml of 1 mg/ml ofPaclitaxel (obtained from Sigma Chemical Co.) and 5 mg/ml of Compound(1) was obtained by mixing 0.1 ml of Compound 1 DMSO stock solution (50mg/ml) and 0.1 ml of Paclitaxel DMSO stock solution (10 mg/ml) anddiluting with 1.8 ml of 20% Cremophore RH40 water solution. The finalformulation for the dosing solution was 10% DMSO, 18% Cremophore RH40and 72% water.

The Dosing Solution (Dosing Volume: 0.01 ml/gram=10 ml/kg) was injectedintravenously into the mice bearing MDA-435 human breast tumor.

Protocol

Group Compounds (Dose) 1 Vehicle Only 2 Paclitaxel (5 mg/kg) 3 Compound(1) (50 mg/kg) 4 Paclitaxel (5 mg/kg) + Compound (1) (25 mg/kg) 5Paclitaxel (5 mg/kg) + Compound (1) (50 mg/kg)Dosing Schedule: 3 Times a Week (Monday, Wednesday, Friday) for 3 Weeks5 Mice were Used For Each GroupResults

FIG. 1 shows the effects of Compound (1) on enhancing anti-tumoractivity of Paclitaxel (Taxol). As can be seen from FIG. 1, Compound (1)significantly enhanced anti-tumor activity of Paclitaxel on human breasttumor MDA-435 in nude mice. FIG. 2 shows the effects of Compound (1) andPaclitaxel on the body weight of nude mice bearing MDA-435 human breasttumor. As can be seen from FIG. 2, Compound (1) significantly enhancedanti-tumor activity of Paclitaxel without increasing toxicity.

Example 13

Compounds (1) and (2) Enhance the Anticancer Activity of Paclitaxel invivo

The protocol described in Example 12 was used to test Compounds (1) and(2) for their ability to enhance the anti-cancer activity of paclitaxelin mice, except as modified as described below.

Protocol

Group Compounds (Dose) 1 Vehicle Only 2 Paclitaxel (2 mg/kg) 3Paclitaxel (5 mg/kg) 4 Compound (1) (80 mg/kg) 5 Compound (2) (80 mg/kg)6 Paclitaxel (2 mg/kg) + Compound (1) (80 mg/kg) 7 Paclitaxel (5mg/kg) + Compound (1) (80 mg/kg) 8 Paclitaxel (2 mg/kg) + Compound (2)(80 mg/kg) 9 Paclitaxel (5 mg/kg) + Compound (2) (80 mg/kg)Dosing Schedule: 3 Times a Week (Monday, Wednesday, Friday) for 3 Weeks5 Mice were Used for Each Group.Results

Average Tumor Volume (mm³) Group on Day 23 % Tumor Growth 1 301.3 100 2259.8 86 3 164.8 55 4 270.0 90 5 305.8 101 6 193.3 64 7 106.2 35 8 148.449 9 60.6 20

Compounds (1) and (2) significantly enhanced anti-tumor activity ofPaclitaxel at both of 2 mg/kg and 5 mg/kg without increasing toxicity.

Example 14

Compound (1)

Enhances the Anticancer Activity of Paclitaxel in vivo

The protocol described in Example 12 was used to test Compound (1) forits ability to enhance the anti-cancer activity of paclitaxel in mice,except modified as described below.

Protocol

Group Compounds (Dose) 1 Vehicle Only 2 Paclitaxel (10 mg/kg) 3 Compound(1) (50 mg/kg) 4 Paclitaxel (10 mg/kg) + Compound (1) (25 mg/kg)Dosing Schedule

-   3 times a week (Monday, Wednesday, Friday) for 3 weeks-   5 mice were used for each group    Results

Average Tumor % Tumor Growth Group Volume [mm³] Inhibition on Day 48 1752.2 — 2 105.4    86% 3 754.9    0% 4 0.59 >99.9%

When 10 mg/kg of Paclitaxel was used, significant anti-tumor activitywas observed. However, after drug treatment (Day 1˜20) terminated, thetumor started growing to become 105 mm³ volume on Day 43. On the otherhand, average tumor volume after treatment of Paclitaxel (10 mg/kg) plusCompound (1) (25 mg/kg) was only 0.59 mm³ with more than 99.9% tumorgrowth inhibition.

Example 15

Compounds (3)-(5) Enhance the Anticancer Activity of Paclitaxel in vivo

The protocol described in Example 12 was used to test Compounds (3)-(5)for their ability to enhance the anti-cancer activity of paclitaxel inmice, except modified as described below.

Protocol

Group Compounds (Dose) 1 Vehicle Only 2 Paclitaxel (5 mg/kg) 3Paclitaxel (5 mg/kg) + Compound (3) (50 mg/kg) 4 Paclitaxel (5 mg/kg) +Compound (4) (100 mg/kg) 5 Paclitaxel (5 mg/kg) + Compound (5) (100mg/kg)Dosing Schedule

-   3 times a week (Monday, Wednesday, Friday) for 3 weeks-   5 mice were used for each group    Results

Group Average % Tumor Growth Inhibition on Day 27 2 19 3 76 4 66 5 79Compounds (3)-(5) demonstrated significant enhancing effects of Taxolanti-tumor activity.

Example 16

Compound (6) Enhances the Anti-Cancer Activity of Paclitaxel in vivo(Human Xenograft Model: Human Breast Carcinoma MDA-435 in Nude Mice)

The protocol described in Example 12 was used to test Compound (6) fortheir ability to enhance the anti-cancer activity of paclitaxel in mice,except modified as described below.

Compound stock solutions were prepared by dissolving the compound in a50:50 mixture of EtOH and Cremophor EL (Polyoxyl 35 Castor Oil, BASF,Germany). This stock solution in 50% EtOH/50% CrEL was sonicated in anultrasonic water bath until all the powder dissolved.

Preparation of Dosing Solution for Compound Administration: The compoundstock solution was diluted 1:10 with D5W (5% Dextrose in Water, AbbottLaboratories, USA).: 1) 2.0 ml of 2.5 mg/ml dosing solution of Compound(6) was prepared by diluting 0.2 ml of a 25 mg/ml Compound Stocksolution with 1.8 ml of 100% D5W; and 2) a dosing solution comprising of1.5 mg/ml of Paclitaxel (obtained from Sigma Chemical Co.) and 2.5 mg/mlof Compound (6) was obtained by mixing 0.2 ml of a 50% EtOH/50% CrELstock solution containing 25 mg/ml of Compound (6) and 15 mg/ml ofPaclitaxel with 1.8 ml of a 100% D5W solution. The final formulation forthe dosing solution was 5% EtOH, 5% CrEL, 4.5% Dextrose, and 85.5%water.

The Dosing Solution (Dosing Volume: 0.01 ml/gram=10 ml/kg) was injectedintravenously into the mice bearing MDA-435 human breast tumor.

Protocol

-   Mice: CD-1 nu/nu female (n=5/group)-   Tumor: MDA-435 (Human breast carcinoma)-   Implantation: 5×10⁶ cells/mouse-   Formulation: 5% Cremophor EL, 5% ethanol, and 4.5% glucose water    solution-   Administration route: intravenous bolus injection-   Dosing schedule: weekly×4

Group Drug Treatment (Dose) 1 Vechicle Only 2 Paclitaxel (15 mg/kg) 3Compound (6) (25 mg/kg) 4 Paclitaxel (15 mg/kg) + Compound (6) (25mg/kg)Results

FIG. 27 shows the effects of Compound (6) on enhancing anti-tumoractivity of Paclitaxel (Taxol). As can be seen from FIG. 26, Compound(6) significantly enhanced anti-tumor activity of Paclitaxel on humanbreast tumor MDA-435 in nude mice. FIG. 28 shows the effects of Compound(6) and Paclitaxel on the body weight of nude mice bearing MDA-435 humanbreast tumor. As can be seen from FIG. 28, Compound (6) significantlyenhanced anti-tumor activity of Paclitaxel without increasing toxicity.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method of treating a subject with cancer, said method comprisingadministering to the subject an effective amount of a compoundrepresented by the following structural formula:

or a pharmaceutically acceptable salt thereof.
 2. A method of treating asubject with cancer, said method comprising administering to the subjectan effective amount of a compound represented by the followingstructural formula:

or a pharmaceutically acceptable salt thereof.
 3. A method of treating asubject with cancer, said method comprising administering to the subjectan effective amount of a compound represented by the followingstructural formula:

or a pharmaceutically acceptable salt thereof.